CN111164068B - Biodegradable compound, lipid particle, composition containing lipid particle, and kit - Google Patents

Abstract

The present invention discloses a method represented by the following formula (1)A compound. Q-CHR ₂ (1) (wherein Q is a nitrogen-containing aliphatic group containing at least 2 tertiary nitrogen atoms and not containing oxygen, and R is an aliphatic group containing a biodegradable group). The compound can be combined with other lipids to reduce aggregation to form lipid particles. Further, the lipid particles may be used in pharmaceutical compositions for delivering active agents to cells.

Description

Biodegradable compound, lipid particle, composition containing lipid particle, and kit

Technical Field

The present invention relates to a biodegradable compound having a structure that degrades in cells, and lipid particles containing the same. The present invention also relates to a composition and a kit for delivering an active agent such as a nucleic acid, which contain the lipid particle.

Background

Various liposomes for use in the treatment of diseases are being studied. Liposomes are microcapsules having a nano-sized particle diameter composed of lipids, in which various compounds and the like can be encapsulated, and which are excellent in biocompatibility and the like, and therefore are ideal materials for selectively delivering therapeutic agents and active agents to target sites in living bodies. Under such a purpose, large unilamellar Liposomes (LUVs) having an average particle diameter of 100nm or more are generally used, and various materials have been developed for the materials constituting the membranes thereof.

Such liposomes can be composed of a single lipid. In this case, for example, a phospholipid having a head and a hydrophobic portion bonded to the head is used as the lipid, and the lipid is associated to form a membrane, so that a microcapsule in which an active agent or the like can be encapsulated is formed. However, in order to impart excellent properties to liposomes, a lipid mixture is generally used to construct liposomes. The lipid mixture contains a combination of a lipid having excellent biodegradability, a lipid that inhibits aggregation of the formed liposome, a lipid having an effect of inhibiting leakage of the inclusion, a lipid having a membrane fusion effect, and the like.

In order to further improve the properties of the liposome, each lipid was studied. For example, medical liposomes for gene transfer are preferred to satisfy high biodegradability, high biocompatibility, high active agent transfer property, and low cytotoxicity, and it is desirable that lipids capable of constituting such liposomes can be used.

As such lipids, various compounds have been developed, and the state of organisms to which they are applied and diseases to be treated are also various. It is desirable to increase the selectable lipid class based on these conditions. In addition, a lipid capable of constituting a liposome having characteristics exceeding those of conventional liposomes has been demanded.

Prior art literature

Patent literature

Patent document 1: japanese patent No. 5893611

Patent document 2: japanese patent No. 6093710

Disclosure of Invention

Problems to be solved by the invention

In view of the above problems, the present embodiment provides novel compounds that are lipids capable of constituting liposomes, lipid particles using the same, and compositions and kits.

Means for solving the problems

The compound of the present embodiment is characterized by being represented by the following formula (1),

Q-CHR ₂ (1)

(in the formula (I),

q is a nitrogen-containing aliphatic group containing more than 2 tertiary nitrogen and no oxygen,

r is each independently C ₁₂ ～C ₂₄ At least one R contains in its main chain or in a side chain a linking group L selected from the group consisting of-C (=o) -O-, -O-C (=o) -O-, -S-C (=o) -, -C (=o) -S-, -C (=o) -NH-, and-NH-C (=o) - ^R )。

The lipid particle of the embodiment is characterized by containing the above-described compound.

The composition of the embodiment is characterized by comprising the lipid particles and a carrier.

The kit of the embodiment further comprises a composition comprising the lipid particle and an introducing agent for introducing the lipid particle into a cell.

Drawings

FIG. 1 is a graph showing the enzymatic activities of examples and comparative examples when a lipid plasmid was used for Jurkat cells.

FIG. 2 is a graph showing enzyme activities when lipid plasmids are used for MCF-7 cells in examples and comparative examples.

FIG. 3 is a graph showing the enzymatic activities of examples and comparative examples when a lipid plasmid was used for Huh-7 cells.

FIG. 4 is a graph showing the enzymatic activity of examples when a lipid plasmid is used for Jurkat cells.

FIG. 5 is a graph showing the enzyme activity of Peripheral Blood Mononuclear Cells (PBMC) using a lipid plasmid according to the example.

FIG. 6 is a graph showing the RNA expression levels when RNA was introduced into Jurkat cells using lipid particles in examples and comparative examples.

FIG. 7 is a graph showing the enzymatic activities of the lipid plasmids used for MCF-7 cells in comparative example 4-1 and example 4-2.

FIG. 8 is a graph showing the enzymatic activities of the lipid plasmids used for Huh-7 cells in comparative example 4-1 and example 4-2.

FIG. 9 is a graph showing the enzymatic activities of Jurkat cells using lipid plasmids for comparison example 4-1 and example 4-2.

FIG. 10 is a graph showing the enzymatic activities of the lipid plasmids used for the MCF-7 cells of comparative example 4-2 and example 4-3.

FIG. 11 is a graph showing the enzymatic activities of the lipid plasmids used for Huh-7 cells of comparative example 4-2 and example 4-3.

Detailed Description

[ definition ]

In the present embodiment, when the numerical value ranges are used to represent the numerical value ranges, these have the same units and include both end points unless otherwise specified. For example, 10 to 25 mol% means 10 mol% or more and 25 mol% or less.

In the present embodiment, "C _x ～C _y "and" C _x "etc. means the number of carbons in the molecule or substituent. For example, C ₁ ～C ₆ Alkyl represents an alkyl group having 1 to 6 carbon atoms. In the present embodiment, haloalkyl means a group in which 1 or more hydrogens in an alkyl group are substituted with halogen such as fluorine, and for example, fluoroaryl means a group in which 1 or more hydrogens in an aryl group are substituted with fluorine.

In the present embodiment, unless otherwise specified, alkyl means: a monovalent group formed by removing one hydrogen from any carbon of an alkane. Also, the term alkyl includes straight-chain or branched alkyl. In addition, cycloalkyl means an alkyl group having a cyclic structure. Groups having a cyclic structure substituted with a linear or branched alkyl group are also referred to as cycloalkyl groups.

In addition, alkenyl refers to: a monovalent group formed by removing one hydrogen from any carbon of an olefin.

In addition, the hydrocarbon group means: a group having 1 or 2 or more valences and containing carbon and hydrogen, and optionally oxygen or nitrogen. The aliphatic group is a hydrocarbon group containing no aromatic ring, and may have any of a chain, branched or cyclic structure, or may be a combination thereof. The aliphatic group may contain an unsaturated bond as long as it is not particularly limited. Further, the aliphatic group may contain hetero atoms such as nitrogen, oxygen, sulfur, selenium, fluorine, chlorine, bromine, and the like, as long as it is not particularly limited. The aliphatic group may be a 1-valent group or a polyvalent group. The aromatic hydrocarbon group contains an aromatic ring, and has an aliphatic hydrocarbon group as a substituent as required.

Tertiary nitrogen means nitrogen to which 3 carbons are bonded. Therefore, tertiary nitrogen constitutes a tertiary amine structure having electron donating properties.

[ biodegradable lipid Compound ]

The compound of the embodiment is a compound suitable as a lipid constituting a liposome. The hydrophobic portion of the polymer has a biodegradable group and functions as a biodegradable lipid compound. The head of the composition does not contain a cationic group, and is characterized by being inhibited from binding to intracellular proteins and having low toxicity when applied to a living body. In addition, when the liposome is composed of the lipid compound, the surface of the liposome is not cationic, so that the damage to cells is reduced, and the rate of introduction of an active agent such as a nucleic acid is increased.

The lipid compound is represented by the following general formula (1). In the following, Q in the formula is sometimes referred to as a head, and R is sometimes referred to as a hydrophobic group.

Q-CHR ₂ (1)

(wherein Q is a nitrogen-containing aliphatic group having 2 or more tertiary nitrogen groups and containing no oxygen,

r is each independently C ₁₂ ～C ₂₄ At least one R contains in its main chain or in a side chain a linking group L selected from the group consisting of-C (=o) -O-, -O-C (=o) -O-, -S-C (=o) -, -C (=o) -S-, -C (=o) -NH-, and-NH-C (=o) - ^R )

One feature of the compounds of the embodiments is that the head Q contains more than 2 tertiary nitrogen and is free of oxygen.

Nitrogen constituting an unsubstituted amino group, a quaternary ammonium or the like may be contained within a range not impairing the effect of the embodiment, and nitrogen other than the tertiary nitrogen is preferably not contained. In addition, the head Q does not contain oxygen. Therefore, the head Q does not contain an oxygen group, a hydroxyl group, a carboxyl group, an alkoxy group, a carboxylate (carboxyl) or the like. The head Q may contain an ionic group, but preferably contains no neutral group of a polar group.

An example of Q is preferably represented by the following general formula (1-Q).

R ^Q1 ₂ N-(CR ^Q2 ₂ ) _q1 -NR ^Q1 -(CR ^Q2 ₂ ) _q2 -＊ (1-Q)

(in the formula (I),

R ^Q1 each independently of the other is an alkyl group,

R ^Q2 each independently is hydrogen or an alkyl group,

R ^Q1 r is R ^Q2 Any two of them may be bonded to form a nitrogen-containing alicyclic ring, q1 is a number of 1 to 4,

q2 is a number from 0 to 4,

representation of and-CHR ₂ Is a bonding position of (a)

Here, the alkyl group is preferably C ₁ ～C ₃ An alkyl group.

In addition, R ^Q1 R is R ^Q2 Any two of them may be bonded to form a nitrogen-containing alicyclic ring. The number of constituent elements of the nitrogen-containing alicyclic ring is not particularly limited, but is preferably 4 to 10, and more preferably 5 to 8. Typically, examples of the nitrogen-containing alicyclic ring include piperidine, piperazine, pyrrolidine, imidazolidine, hexamethyleneimine, homopiperazine, heptamethyleneimine, and the like.

Such Q may have, for example, the following structure.

[ chemical 1]

(wherein, the formula is represented by the general formula-CHR ₂ Is a bonding position of (a)

The compounds of the embodiments have a head-bonded-CHR ₂ . Here, R represents a hydrophobic group, and two R may be the same or different. Hydrophobic groups typically contain relatively long hydrocarbon chains. And a part of the polymer contains a linking group including a carboxylate or the like, specifically, the composition comprises a compound selected from the group consisting of-C (=O) -O-, -O-C (=O) -O-, -S-C (=O) -, and-C (=o) -S-, -C (=o) -NH-and-NH-C (=o) -linking groups. When the compound of the embodiment is used in a liposome, these linking groups function as biodegradable groups.

An example of the preferable hydrophobic group R can be represented by the following formula (1-R).

-L ^R1 -C(＝O)-O-L ^R2 (1-R)

(in the formula (I),

L ^R1 is an alkylene group, and is preferably an alkylene group,

L ^R2 alkenyl group)

L ^R1 L and L ^R2 The branched structure and the cyclic structure may be used, and in the case of having a branched structure, the number of side chains is preferably small, and most preferably straight-chain structure is used.

More specifically, L ^R1 L and L ^R2 It is preferably represented by the following formulas (1-R1) and (1-R2).

-(CH ₂ ) _r1 - (1-R1)

-CH ₂ -CH＝CH-(CH ₂ ) _r2 -H (1-R2)

(in the formula (I),

r1 is a number from 1 to 10,

r2 is a number from 1 to 10)

Here, in order to make the hydrophobic group exhibit sufficient hydrophobicity, R1 is preferably a number of 4 to 8, and the longest molecular chain contained in the hydrophobic group R is preferably 8 atoms or more.

Each part of the compound of the embodiment has the structure described above, and the compound of the embodiment preferably has the structures represented by the following formulas (1-01) to (1-21).

[ chemical 2-1]

[ chemical 2-2]

[ chemical conversion 2-3]

Of these, (1-01) and (1-02) are particularly preferred because they exhibit excellent properties when used in liposomes.

[ method for producing Compound ]

The compounds of the present invention may be manufactured by any method. For example, the compounds (1-01) and (1-02) can be produced according to the following process charts.

[ chemical 3]

Synthesis of Compound (1-01)

[ chemical 4-1]

Synthesis of Compound (1-02) (1)

[ chemical 4-2]

Synthesis of Compound (1-02) (2)

[ lipid particles ]

According to an embodiment, a lipid particle is provided. The typical examples of the lipid particles include, but are not limited to, liposomes, and lipid complexes formed by complexing with nucleic acids and the like. In addition, the liposome may be any one of unilamellar liposomes and multilamellar liposomes.

The lipid particle of the embodiment contains the compound represented by the above formula (1). In addition, it is desirable to further contain membrane-forming lipids and lipids that reduce aggregation.

The membrane-forming lipid may be any lipid that is generally used for liposomes. The lipid is preferably a lipid having excellent biodegradability.

Specific examples of such membrane-forming lipids include diacyl phosphatidylcholine, diacyl phosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, cerebroside, and the like. In the embodiment, the lipid used for forming the membrane of the lipid particle may be appropriately selected in consideration of the size of the liposome of interest, the stability of the liposome in the living body, and the like. Of these, diacyl phosphatidylcholine and diacyl phosphatidylethanolamine are preferred. The length of the hydrocarbon chain of the acyl group contained in the lipid is preferably 10 to 20. The hydrocarbon chain may be a saturated hydrocarbon group or an unsaturated hydrocarbon group.

As such a lipid forming a film, various substances are known, and preferable examples thereof include 1, 2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE), 1, 2-stearoyl-sn-glycerol-3-phosphatidylethanolamine (DSPE), 1, 2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (POPC), 1, 2-di-O-octadecyl-3-trimethylammonium propane (DOTMA), 1, 2-dioleoyl-3-dimethylammonium propane (DODAP), 1, 2-dimyristoyl-3-dimethylammonium propane (14:0 DAP), 1, 2-dipalmitoyl-3-dimethylammonium propane (16:0 DAP), 1, 2-distearoyl-3-dimethylammonium propane (18:18), DAP), 4- (carboxy) -2-dioleoyl-3-trimethylammonium propane (DOTMA), 1, 2-dioleoyl-3-dimethylammonium propane (DODAP), 1, 2-dioleoyl-3-dimethylammonium propane (DOPA), 2-dP), 2-dioleoyl-3-dimethylammonium propane (DOPA) 1, 2-dioleoyl-sn-glycerol-3-phosphate-L-serine (DOPS), cholesterol, and the like. Further, DOPE, DOTAP or cholesterol is more preferable, and a combination of DOPE and cholesterol, a combination of DOTAP and cholesterol, and a combination of DOPE and DOTAP and cholesterol is particularly preferable. These can exert a membrane fusion effect in addition to a function of forming a membrane such as a liposome.

The aggregation-reducible lipids used in the embodiments exert aggregation-containing functions among particles in the preparation of lipid particles. As such a lipid, various substances are known, and any of these substances can be selected for use in the lipid particles of the embodiment. Examples of such lipids include polyethylene glycol (PEG) modified lipids, polyamide oligomers derived from omega-amino (oligoethylene glycol) alkanoic acid monomers (U.S. Pat. No. 6,320,017), monosialogangliosides, and the like. More specifically, ATTA lipids such as ATTA8-DPSE listed in U.S. Pat. No. 6,320,017 and polyethylene glycol lipid conjugates described in U.S. Pat. Nos. 5,820,873, 5,534,499 and 5,885,613 can be used.

In forming the lipid particle, the PEG-modified lipid may form an anchored lipid moiety on the surface of the lipid particle. Examples of such PEG-modified lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-ceramide conjugates (e.g., C14 PEG-Cer or C20 PEG-Cer described in Japanese patent No. 3920330), PEG-modified dialkylamines, PEG-modified 1, 2-diacyloxopropane-3-amines, PEG-modified diacylglycerols (e.g., 1, 2-dimyristoyl-sn-glycerol-methoxypolyethylene glycol; PEG-DMG), and PEG-modified dialkylglycerols. Among them, PEG-modified diacylglycerols and PEG-modified dialkylglycerols are particularly preferable.

When a bulky modification group such as PEG is bound to the lipid surface, the binding of the modification group to the lipid particle may affect the stability of the lipid particle or liposome. For example, U.S. patent No. 5,820,873 discloses that the characteristics of the length of the acyl chain, the saturation of the acyl chain, and the size of the steric head group in PEG-modified lipids affect the stability of the lipid particle. Accordingly, the desired lipid particle can be obtained by adjusting these properties. For example, the lipid particles may be disappeared more quickly by shortening the modifying groups in the PEG-modified lipid, or the residence time in plasma may be prolonged by lengthening the modifying groups, or the like. As a result, delivery of lipid particles to target tissues may sometimes be improved.

The lipid particles may also contain other lipids. Such other lipid may be arbitrarily selected from substances commonly used for lipid particles and used. For example, lipids with relatively low toxicity may be combined in order to modulate toxicity. In addition, lipids having a specific structure may be combined in order to introduce a functional group for binding a ligand into the lipid particle.

Further, when lipid particles are used as liposomes, sterols such as cholesterol may be contained as lipids for inhibiting leakage of the inclusion. Further, a targeting agent may be coupled to the lipid particle. The coupling method in this case may be any conventionally known method.

The lipid particle of the embodiment preferably further contains a compound represented by the following formula (2) and having at least one biodegradable group selected from the group consisting of a carboxylic acid ester bond, a thiocarboxylic acid ester bond, a dithiocarboxylic acid bond, an amide bond, a urethane bond, a carboxydioxy bond and a urea bond in the structure,

P-[X-W-Y-W’-Z] ₂ (2)

(in the formula (I),

p is an alkyleneoxy group having 1 or more ether linkages in the main chain,

x is each independently a 2-valent linking group comprising a tertiary amine structure,

w is each independently C ₁ ～C ₆ An alkylene group,

y is each independently a 2-valent linking group selected from the group consisting of single bonds, ether bonds, carboxylic ester bonds, thiocarboxylic ester bonds, amide bonds, urethane bonds, and urea bonds,

w' are each independently a single bond or C ₁ ～C ₆ Alkylene groups, Z are each independently a fat-soluble vitamin residue, a sterol residue or C ₁₂ ～C ₂₂ Aliphatic hydrocarbon groups).

The compound represented by the formula (2) is also a lipid compound having biodegradability. By combining the compound of formula (2) with the compound of formula (1), a novel function can be exhibited. For example, when the compound of formula (2) is applied to a liposome, the amount of nucleic acid entrapped can be increased, and when the compound of formula (1) and the compound of formula (2) are applied to a liposome, they can be more easily applied to gene therapy, nucleic acid therapy, genomic diagnosis, and the like.

One feature of the compound of formula (2) is that P in formula (2) contains an ether bond. That is, P contains at least one oxygen, and the oxygen is bonded to two carbons.

The number of oxygen contained in P is not particularly limited, and preferably contains 1 to 2 oxygen atoms. The number of carbon atoms contained in P is not particularly limited, and the number of carbon atoms of the hydrocarbon chain contained in P is preferably 1 to 3, and the total number of carbon atoms contained in P is preferably 3 to 8. Preferable examples of P include the following.

-(CH ₂ ) ₂ -O-(CH ₂ ) ₂ -

-(CH ₂ ) ₂ -O-(CH ₂ ) ₂ -O-(CH ₂ ) ₂ -

-(CH ₂ ) ₂ -O-O-(CH ₂ ) ₂ -

-(CH ₂ ) ₃ -O-(CH ₂ ) ₂ -O-(CH ₂ ) ₃ -

-(CH ₂ ) ₂ -O-CH ₂ -O-(CH ₂ ) ₂ -

By having such a structure, the degree of freedom of the three-dimensional structure is improved. When this compound is used for the constitution of a liposome, oxygen contained in an ether bond forms a hydrogen bond with a nucleic acid or the like to be combined, and thus the amount of inclusion of the nucleic acid or the like increases.

In addition, X is a 2-valent linking group containing a tertiary amine structure, and is preferably selected from the group consisting of methylimino, 1, 2-pyrrolidinediyl, and 1, 3-pyrrolidinediyl. When this compound is used for liposome formation, high cell membrane permeability can be exhibited by the tertiary amine structure.

In the formula (2), the moiety-W-Y-W' -Z constitutes a hydrophobic moiety. The hydrophobic portion contains a biodegradable group. Here, the biodegradable group is selected from the group consisting of a carboxylic ester bond (-C (=o) -O-), a thiocarboxylic ester bond (-C (=o) -S-), a dithiocarboxylic ester bond (-C (=s) -S-), an amide bond (-C (=o) -NH-), a urethane bond (-NH-C (=o) -O-), a carboxyl dioxy bond (-O-C (=o) -O-) and a urea bond (-NH-C (=o) -NH-).

The biodegradable group may be contained in Z in addition to the structure as Y. That is, Z is a group derived from a fat-soluble vitamin or sterol, and the structure thereof may contain a carboxylic acid ester group or the like. The biodegradable group may be contained in both Z and Y, or may contain 2 or more biodegradable groups.

Y and W' are 2-valent groups linking W and Z.

Y is selected from the group consisting of single bonds, ether bonds, carboxylic ester bonds, thiocarboxylic ester bonds, and amidesA bond, a urethane bond, and a urea bond. In addition, W' is a single bond or C ₁ ～C ₆ An alkylene group.

These may be single bonds without elements. On the other hand, when Z does not contain a biodegradable group, Y contains a biodegradable group.

Z is a fat-soluble vitamin residue, a sterol residue or C ₁₂ ～C ₂₂ Aliphatic hydrocarbon groups. Of these, a fat-soluble vitamin residue or a sterol residue is preferable, and a fat-soluble vitamin residue is more preferable.

The fat-soluble vitamin residue is a group derived from a fat-soluble vitamin. Examples of the fat-soluble vitamins include retinol, retinal, ergosterol, 7-dehydrocholesterol, ergocalciferol, cholecalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol and tocotrienol. These fat-soluble vitamins have hydroxyl groups at the ends. An example of the fat-soluble vitamin residue is a group obtained by removing hydrogen from the hydroxyl group of the above. In addition, groups derived from fat-soluble vitamin derivatives may also be used. The fat-soluble vitamin derivative is a compound in which a hydroxyl group contained in a fat-soluble vitamin is replaced with a thiol group, a carboxyl group, a thiocarboxyl group or a dithiocarboxyl group. These fat-soluble vitamin residues have-S-, -C (=o) -O-, -C (=o) -S-, or-C (=s) -S-, at the terminal. Of the fat-soluble vitamin residues, groups derived from retinol (vitamin a), tocopherol (vitamin E) or carboxylic acid derivatives thereof are particularly preferred.

Sterol residues are groups derived from sterols. Examples of sterols include cholesterol, stigmasterol, β -sitosterol, lanosterol, and ergosterol. One example of a sterol residue is a group obtained by removing hydrogen from the hydroxyl group of these sterols. The sterol residue may have the same terminal group as the group derived from the fat-soluble vitamin derivative. Particularly preferred sterol residues are groups derived from sterols, cholesterol or carboxylic acid derivatives thereof.

C ₁₂ ～C ₂₂ The aliphatic hydrocarbon group may be either a straight chain or branched chain, or may have a cyclic structure. In addition, the greaseThe group hydrocarbon group may contain an unsaturated bond, and in the case of containing it, it usually contains 6 or less, preferably 3 or less unsaturated bonds. The carbon number of the aliphatic hydrocarbon group is preferably 12 to 18, more preferably 13 to 17.

Among these Z groups, those containing a structure that absorbs ultraviolet rays are preferable. Specifically, it preferably contains a cyclohexene structure. Thus, by having a structure that absorbs ultraviolet light, degradation of lipid particles containing the compound as a component due to light can be reduced, and when it is necessary to analyze the behavior of lipid particles, the analysis can be easily performed.

The compound of formula (2) contains two [ X-W-Y-W '-Z ], each X, W, Y, W' and Z is independent and may be the same or different, and preferably the same and the structure of the compound is symmetrical.

Each part of the compound of the embodiment has the structure described above, and the compound of the embodiment preferably has the structures represented by the following formulas (2-01) to (2-12).

[ chemical 5-1]

[ chemical 5-2]

[ chemical conversion 5-3]

Of these, (2-01) to (2-04) are particularly preferable because they can exhibit excellent properties when used in combination with the compound of formula (1) for use in liposomes.

The compound of formula (2) can be produced, for example, according to the following process scheme.

[ chemical 6]

The lipid particles may be constituted by combining these lipids, and the mixing ratio of each lipid constituting the lipid particles may be adjusted according to the purpose, and thus is not limited. However, in general, the lipid compounds represented by the formulas (1) and (2) are blended in an amount of 25 to 75 mol% in total, and 1 to 10 mol% in total, preferably 30 to 60 mol% in total, 30 to 65 mol% in total, and 1 to 10 mol% in total, for example, 2.5 mol% in total, based on the total number of the lipids used in the lipid particles. Here, the balance between the compound of formula (1) and the membrane-forming lipid is important, and the rate of introduction of the active agent cannot be sufficiently increased by the presence of only one of them. Therefore, the compounding ratio of the compound of formula (1) or (2) to the membrane-forming lipid is preferably 1 on a molar basis: 0.5 to 1:3, more preferably 1:0.75 to 1:2.1.

The compound represented by the formula (1) or (2) also functions as a membrane-forming lipid, but in the embodiment, "membrane-forming lipid" does not include the compound of the formula (1) or (2).

The lipid particle of an embodiment may further contain an active agent. In embodiments, an active agent refers to a substance that can impart a particular effect on a cell, tissue, organ, or subject. The specific effect may be any of biological, physiological or cosmetic effects. By using the lipid particles of the embodiments, various active agents can be delivered to a target site within an organism. The active agent may be encapsulated in the lipid particles, bound to the lipid surface on the outside or inside, or disposed in the lipid layer.

Typical examples of the active agent are nucleic acids, and examples thereof include nucleic acids selected from the group consisting of plasmids, oligonucleotides, polynucleotides, small interfering RNAs (siRNAs), microRNAs (miRNAs), DNAs, aptamers, and ribozymes. In addition, antisense oligonucleotides, antago-mir, aDNA, plasmids, ribosomal RNA (rRNA), transfer RNA (tRNA), nuclear low molecular RNA (snRNA), mRNA, and the like can also be used. Different kinds of DNA and RNA may be used in combination.

As the miRNA, a miRNA obtained by ligating 17 to 25 nucleotide units can be used. In a more preferred embodiment, the nucleic acid is an oligonucleotide of 15 to 50 or 20 to 30 nucleotide units linked. The siRNA may have a double-stranded region, for example, containing 16 to 30 nucleotide units. In another embodiment, the nucleic acid is an immunostimulatory oligonucleotide, decoy oligonucleotide, supermir, miRNA mimic, or miRNA inhibitor. Supermir refers to a single-, double-or partially double-stranded oligomer or polymer of RNA or deoxyribonucleic acid DNA, or both, or modifications thereof, having a nucleotide sequence substantially identical to that of a miRNA, and being antisense to its target. miRNA mimics represent a group of molecules that can be used for the purpose of mimicking the gene silencing ability of 1 or more mirnas. Thus, the term "miRNA mimic" refers to a synthetic non-coding RNA that can enter the RNAi pathway and regulate gene expression (i.e., miRNA mimics cannot be purified from a supply of endogenous miRNA).

When the nucleic acid is combined with the lipid particle, the form of the nucleic acid is not particularly limited. The nucleic acid may be, for example, single-stranded DNA or RNA, double-stranded DNA or RNA, or a DNA-RNA hybrid. As an example of double-stranded RNA, siRNA can be cited. Examples of the single-stranded nucleic acid include antisense oligonucleotides, ribozymes, miRNAs, and triplex oligonucleotides.

When the lipid particle of the embodiment contains a nucleic acid, it may further contain a compound that binds to the nucleic acid. Examples of such a compound include basic proteins and basic peptides, and preferably include protamine, histone and salts thereof. For example, histones and salts thereof have the property of binding to nucleic acids and folding nucleic acid molecules. In addition, protamine has the property of binding to and involving nucleic acid. Thus, these compounds are effective in encapsulating nucleic acids in lipid particles.

In addition, the lipid particle of the embodiment may further contain a compound that regulates expression of nucleic acid in a cell. By controlling the expression of nucleic acids in cells, the effect of visualization and apoptosis on the cells to which the liposomes are delivered can be achieved, and is therefore preferred. Examples of such compounds include retinoic acid, cyclic adenosine monophosphate (cAMP), and ascorbic acid.

The lipid particle according to the embodiment may contain lipoproteins, apolipoproteins, and the like.

As active agents, other therapeutic agents may also be used. Specific examples of the therapeutic agent that can be used include peptides, polypeptides, cytokines, growth factors, apoptosis-inducing factors, differentiation-inducing factors, cell surface receptors and ligands thereof, hormones, and the like. More specifically, the therapeutic agent may be exemplified by an anti-inflammatory compound, an antidepressant, a stimulant, an analgesic, an antibiotic, a contraceptive, an antipyretic, a vasodilator, an angiogenesis inhibitor, a cell vascular action agent (cytovascular agents), a signal transduction inhibitor, a cardiovascular agent, an antitumor agent, a hormone, and a steroid.

When the active agent is combined with the lipid particle, the active agent is preferably introduced into the lipid particle at a higher rate of introduction. In addition, it is also preferable that the cell death rate caused by cytotoxicity depending on lipid properties is low. When nucleic acid is introduced using conventionally known lipid particles, the introduction rate is generally low and the proportion of cell death due to cytotoxicity is also high. In contrast, when the lipid particle according to the present embodiment is used, the nucleic acid introduction rate can be increased and cell death can be reduced. Specifically, the conventional lipid particles have an introduction rate of about 10%, and cell death by electroporation is 60 to 70%, whereas in the case of using the lipid particles of the embodiment, the introduction rate is 70% or more, and cell death by electroporation is reduced to 30% or less.

The lipid particles of the embodiments may be formed into any size according to purposes. However, in the case where the lipid particles of the embodiment are to be used for pharmaceutical use, the lipid particles are usually formed into particles of a nano-scale size. Specifically, the average particle diameter of the lipid particles of the embodiment is usually 50nm to 300nm, preferably 50nm to 200nm. The size of the lipid particles may be adjusted by any method. For example, the lipid particles may be reduced by ultrasonic treatment. The size may be adjusted by classifying the lipid particles through a polycarbonate film or a ceramic film. In the embodiment, the average particle diameter of the lipid particles can be measured by, for example, a Zetasizer using a dynamic light scattering method.

In addition, the in vivo half-life (t 1/2) of the lipid particles of the embodiments is generally less than 3 hours, preferably less than 2 hours, and particularly preferably less than 1 hour. Herein, in vivo half-life refers to half-life in, for example, liver, spleen or plasma. In an embodiment, since the compound of formula (1) constituting the lipid has a biodegradable group, the half-life is, for example, less than 10% compared to a lipid particle constituted of a lipid containing no biodegradable group.

[ method for producing lipid particles ]

The lipid particles of the embodiment can be produced by any conventionally known method. As methods for producing lipid particles and liposomes, the method of back (Bangham), the organic solvent extraction method, the surfactant removal method, the freeze thawing method, and the like are known, and these methods can be used. In addition, for example, by introducing the compound represented by the formula (1) and a film-forming lipid or reducing the amount of aggregated lipid into an organic solvent such as alcohol, and adding an aqueous buffer, lipid particles can be spontaneously formed. The active agent can be introduced into the lipid particles by previously combining the active agent with the aqueous buffer.

[ use of lipid particles ]

The lipid particles of the present embodiments can be used to deliver an active agent to a cell. In particular, delivery of an active agent such as a nucleic acid to a cell is used in all fields such as genetic engineering, production of recombinant proteins, and medical techniques known as gene therapy and cellular diagnosis. In one embodiment, a composition for delivering an active agent to a cell is provided, comprising the lipid particle of the embodiment and a carrier. In another embodiment, a lipid particle of an embodiment for delivering an active agent to a cell is provided. In another embodiment, a method for delivering an active agent to a cell is provided, comprising: the lipid particles of the embodiment containing the active agent are contacted with cells (e.g., the lipid particles are administered to a subject). In another embodiment, there is provided the use of a lipid particle according to any one of claims 9 to 22 for delivering an active agent to a cell. In one embodiment, the lipid particle contains both the compound of formula (1) and the compound of formula (2), and the molar ratio of the content of the compound of formula (2) to the content of the compound of formula (1) is preferably less than 1. In one embodiment, the cells are tumor cells. The subject is preferably an animal, more preferably a mammal, and most preferably a human in need of such treatment. These uses will be described in detail later.

[ composition ]

The lipid particle of the present embodiment may be used in the form of a composition. For example, a composition containing the lipid particle and the carrier according to the present embodiment is provided. Such compositions may also be used for pharmaceutical purposes.

The carrier may be optionally used from conventionally known carriers, and examples thereof include saline such as water and physiological saline, glycine aqueous solution, and buffer. In addition to these carriers, glycoproteins such as albumin, lipoprotein, apolipoprotein, and globulin may be combined for the purpose of improving stability and the like.

The compositions of the embodiments may be prepared by standard methods. As the carrier, physiological saline is generally used. For compositions containing saline or other saline carriers, it is preferred to add the carrier after formation of the lipid particles. Therefore, generally, after combining the lipid particles and the active agent such as nucleic acid, the composition is replaced or diluted with a pharmaceutically acceptable carrier such as physiological saline.

The composition of the embodiment may contain an auxiliary agent as needed. For example, in the case of pharmaceutical use, the pharmaceutical composition may be brought closer to the physiological state by containing pharmaceutically acceptable auxiliaries, such as pH adjusting agents, buffers, tonicity adjusting agents, and the like, as auxiliaries. Examples of the auxiliary agents that function as such an action include sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, and hydroxyethylpiperazine ethane sulfonic acid (HEPES). In addition, the composition of the embodiment may contain a lipid protecting agent for improving storage stability. Examples of such a protective agent include lipophilic radical quenchers such as α -tocopherol which inhibit damage caused by radicals, and water-soluble chelating agents such as feverfew which inhibit peroxidative damage of lipids.

In addition, the above-described active agents and the like may also be added to the composition. The active agent may be the same as or different from the active agent combined in the lipid particle. In addition, a compound that binds to a nucleic acid or a compound that regulates expression of a nucleic acid may be added to the composition.

The concentration of the lipid particles contained in the composition of the embodiment is not particularly limited, and the content of the lipid particles contained in the composition is usually 0.01 to 30% by mass, preferably 0.05 to 10% by mass. The concentration of the lipid particles may be appropriately selected according to the purpose.

The composition of the embodiment may be sterilized by a conventionally known method. The sterilized composition may be packaged as a directly administrable preparation, or may be packaged after drying. The dried composition may be formulated for administration by combining with a sterile aqueous solution immediately prior to administration.

The composition of the embodiments may also be in the form of a kit. The kit of the embodiment contains the above lipid particles and an introducing agent for introducing the above lipid particles into cells, but the form thereof is arbitrary. Examples include: a kit in which a dispersion of lipid particles containing no active agent in a carrier and an active agent are contained in separate containers, a kit in which dried lipid particles, an active agent, and a carrier are contained in separate containers, and the like. Further, the dried lipid particles or lipid particle dispersion and the active agent may be prepared as separate products, and the user may select each product according to the purpose.

The kit may be combined with a reagent used for introducing nucleic acid.

[ method of Using pharmaceutical composition ]

When the lipid particles of the embodiments are used for pharmaceutical use, the compositions can be used to treat or diagnose various diseases in humans or animals. For example, by employing a therapeutic agent as an active agent in combination with a lipid particle, the therapeutic agent can be delivered to a target cell for treatment.

For example, various nucleic acids may be delivered to cells in contact therewith for disease prevention or treatment. Examples of such nucleic acids include oligonucleotides, siRNA, plasmids, antisense nucleic acids, and ribozymes. The lipid compounds of these embodiments can efficiently and rapidly ingest these nucleic acids. For example, it has been difficult to safely introduce RNA into lipid particles in a short period of time, but the introduction can be easily performed by using the lipid compound according to the embodiment.

By appropriately combining a lipid compound for preparing lipid particles and a film-forming compound, more efficient cell targeting can be performed. Specifically, the combination of the compound (1-01), the compound (2-01), DOPE and cholesterol can be suitably used for delivery to liver cancer cells, the combination of the compound (1-01) or the compound (1-02), DOTAP and cholesterol can be suitably used for delivery to T-cell leukemia cells, and the combination of the compound (1-01), the compound (2-01), DOTAP, DOPE and cholesterol can be suitably used for delivery to breast cancer cells.

In addition, by appropriately combining a lipid compound for producing lipid particles and a film-forming compound, diagnosis, treatment, and prevention can be performed more effectively. Specifically, the combination of the compound (1-01), the compound (2-01), DOPE and cholesterol is suitable for diagnosis, treatment and prevention of liver cancer, the combination of the compound (1-01) or the compound (1-02), DOTAP and cholesterol is suitable for diagnosis, treatment and prevention of T-cell leukemia, and the combination of the compound (1-01), the compound (2-01), DOTAP, DOPE and cholesterol is suitable for diagnosis, treatment and prevention of breast cancer.

In addition, delivery of nucleic acids may be performed either in vitro or in vivo. As a method of in vivo administration, the pharmaceutical composition is preferably administered non-orally, i.e., intra-articular administration, intravenous administration, intraperitoneal administration, subcutaneous administration, or intramuscular administration. Intravenous or intraperitoneal administration of the pharmaceutical composition may be performed by bolus injection.

Alternatively, the pharmaceutical formulation of the embodiments may be applied directly to the target tissue, thereby bringing the pharmaceutical composition into contact with the target tissue. In addition, the administration to the meninges and the like can be performed by instillation, and the administration can be performed using an endoscopic tool.

In a particular embodiment, the treatment with the pharmaceutical composition is generally carried out at physiological temperature (about 37 ℃) for a period of time ranging from 1 to 24 hours, preferably from 2 to 8 hours. In an in vitro application, the cells to be treated are not particularly limited. For example, it may be a vertebrate cell, a non-vertebrate cell or a plant cell. However, in a preferred embodiment, the cells are animal cells, more preferably mammalian cells, most preferably human cells.

Examples

Synthesis example 1 Synthesis of Compound (1-01)

The synthesis of the compound (1-01) was carried out according to the above-mentioned production process. Specifically, the following operations are performed.

Step 1

Magnesium (17.38 g, 714.96mol, 4.4 eq.) diethyl ether (165 mL) and iodine (7 mg) were added to a 500mL flask under an argon atmosphere. After a few drops of 9-bromonon-1-ene (100.00 g, 487.47mol, 3 eq.) were added dropwise at room temperature, the mixture was added dropwise under reflux for 2 hours. After aging overnight at room temperature, the Grignard reagent was transferred to a dropping funnel while washing with diethyl ether (40 mL). The Grignard reagent was added dropwise to a 1000mL four-necked flask containing ethyl formate (12.04 g, 162.49mol, 1 eq.) and diethyl ether (165 mL) at 0℃for 1.5 hours.

After reacting at room temperature for 1 hour, acetone (100 mL) was added, followed by water (200 mL) and 10% aqueous sulfuric acid (267 mL) to separate the solutions. The aqueous layer was extracted with diethyl ether (300 mL) and the organic layer was dried over sodium sulfate. The crude product (72.1 g) obtained by filtration and concentration was purified by column chromatography (silica gel 721g, development: hexane → 3% ethyl acetate/97% hexane) to obtain 41.1g (yield 98%) of intermediate (1-01-1) as a white solid.

Step 2

Intermediate (1-01-1) (41.1 g, 146.53mmol, 1 eq.) was dissolved in methylene chloride (330 mL) under an argon atmosphere, and then charged into a 1000mL flask, and triethylamine (59.31 g, 586.12mmol, 4 eq.) and 4-dimethylaminopyridine (1.79 g, 14.65mmol, 0.1 eq.) were added. Methanesulfonyl chloride (33.57 g, 293.06mmol, 2 eq.) was added dropwise at-5 ℃. After stirring at room temperature for 1 hour, ice water (17.6 mL) was quenched. Then, the mixture was washed with 1N hydrochloric acid (30 mL), water (300 mL) and saturated brine (300 mL), and dried over sodium sulfate. Filtration and concentration gave intermediate (1-01-2) 49.6g (yield 94%) as an orange oil.

Step 3

DMF (300 mL) and sodium cyanide (13.56 g, 276.65mmol, 2 eq.) were added to a 1000mL flask under argon. Intermediate (1-01-2) (49.6 g, 138.32mmol, 1 eq.) dissolved in DMF (200 mL) was added, warmed to 55℃and reacted overnight. The reaction mixture was allowed to return to room temperature, diluted with water (500 mL), and extracted repeatedly 3 times with ethyl acetate (800 mL). The extracted organic layer was washed with water (500 mL) and saturated brine (500 mL), and dried over sodium sulfate. The crude product (84.3 g) obtained by filtration and concentration was purified by column chromatography (silica gel 1012 g: development: hexane → 5% ethyl acetate/95% hexane) to obtain intermediate 3.1 g (yield 70%) as a pale yellow oil.

Step 4

To a 2000mL flask was added intermediate (1-01-3) (28.1 g, 97.06mmol, 1 eq.) and hexane (280 mL) under an argon atmosphere. 1M diisobutylaluminum hydride (DIBAL-H) in n-hexane (194.13 mL, 194.13mmol, 2 eq.) was added dropwise at-70℃and stirred at room temperature for 30 minutes. Cooled to 0℃with ice and quenched with methanol (14 mL). To the reaction mixture was added a saturated aqueous ammonium chloride solution (1200 mL), and the mixture was stirred for 20 minutes, followed by addition of 10% sulfuric acid (450 mL) and separation. Then, the mixture was extracted 2 times with diethyl ether (500 mL). The extracted organic layer was washed with a saturated aqueous solution (500 mL) of sodium hydrogencarbonate and saturated brine (500 mL), and dried over sodium sulfate. Filtration and concentration gave intermediate (1-01-4) 25.3g (yield 89%) as a yellow oil.

Step 5

To a 1000mL flask was added intermediate (1-01-4) (25.3 g, 86.5mmol, 1 eq.) and methanol (253 mL). Sodium borohydride (1.16 g, 30.27mmol, 0.35 eq.) was added little by little at 0deg.C and stirred overnight at room temperature. Acetic acid (7 mL) was added to the reaction until pH4 was reached. Water (160 mL) was added and extracted 3 times with dichloromethane (400 mL) and the organic layer was dried over sodium sulfate. The crude product (30.3 g) obtained by filtration and concentration was purified by column chromatography (silica gel 304g development: 5% ethyl acetate/95% hexane) to obtain 22.13g (yield 87%) of intermediate (1-01-5) as a pale yellow oil.

Step 6

Intermediate (1-01-5) (22.13 g, 75.14mmol, 1 eq.) was dissolved in methylene chloride (220 mL) in a 1000mL flask under argon atmosphere, and tetrabromomethane (29.90 g, 90.17mmol, 1.2 eq.) was added. Triphenylphosphine (29.56 g, 112.71mmol, 1.5 eq.) in dichloromethane (63 mL) was added dropwise at 0 ℃. After stirring at room temperature for 1 hour, the reaction solution was concentrated, and the obtained crude product (21.3 g) was purified by column chromatography (silica gel 200g development: hexane) to obtain 14.5g (yield 54%) of intermediate (1-01-6) as a colorless transparent oil.

Step 7

To a 1000mL flask was added intermediate (1-01-6) (5 g, 13.99mmol, 1 eq.) which was dissolved in methyl chloride (230 mL) and acetonitrile (230 mL), and ruthenium (III) chloride (145 mg, 0.69mmol, ru=40%) was added. Sodium periodate (29.92 g, 139.89mmol, 10 eq.) dissolved in water (115 mL) was added dropwise below 10 ℃ and stirred overnight at room temperature. After the completion of the reaction, water (230 mL) was added thereto, and the mixture was separated. The aqueous layer was extracted with dichloromethane (100 mL. Times.2), and to the combined organic layers was added saturated brine (230 mL) and 3% sodium sulfide was added dropwise until discoloration occurred. 1M hydrochloric acid was added until it became acidic, and the mixture was separated. The organic layer was dried over sodium sulfate. The crude product (14.7 g) obtained by filtration and concentration was purified by column chromatography (silica gel 300 g: chloroform- > 2% methanol/98% chloroform) to obtain 2.73g (yield 49%) of the intermediate (1-01-7) as a pale yellow oil.

Step 8

In a 100mL flask, intermediate (1-01-7) (2.73 g, 6.94mmol, 1 eq.) was dissolved in dichloromethane (45 mL), and cis-2-nonen-1-ol (2.41 g, 16.93mmol, 2.44 eq.), 4-dimethylaminopyridine (85 mg, 0.69mmol, 0.1 eq.), N-diisopropylethylamine (4.39 g, 34.01mmol, 4.9 eq.) were added. Then, 1- (3-dimethylaminopropyl) -3-ethyl-carbodiimide hydrochloride (3.25 g, 16.93mmol, 2.44 eq.) was added and stirred overnight at room temperature. After completion of the reaction, the reaction mixture was diluted with dichloromethane (45 mL), washed with water (45 mL), 1M hydrochloric acid (90 mL), a saturated aqueous solution of sodium hydrogencarbonate (90 mL) and saturated brine (90 mL) in this order, and the organic layer was dried over sodium sulfate. The crude product (3.7 g) obtained by filtration and concentration was purified by column chromatography (silica gel 37g development: hexane → 5% ethyl acetate/95% hexane) to obtain 1.62g (yield 36%) of intermediate (1-01-8) as a yellowish oil.

Step 9

Intermediate (1-01-8) (1.62 g, 2.52mmol, 1 eq.) was dissolved in THF (30 mL) in a 50mL autoclave, and N, N' -trimethylethylenediamine (5.16 g, 50.48mmol, 20 eq.) and potassium carbonate (1.26 g, 9.09mmol, 3.6 eq.) were added. The temperature was raised to 55℃and the reaction was carried out for 6 days. After completion of the reaction, the reaction mixture was allowed to return to room temperature, diluted with methylene chloride (60 mL), and water (30 mL) was added thereto to separate the mixture. The aqueous layer was extracted 3 times with dichloromethane (20 mL) and the combined organic layers were dried over sodium sulfate. The crude product (2.3 g) obtained by filtration and concentration was purified by column chromatography (silica gel 25 g: chloroform- > 5% methanol/95% chloroform) to obtain 1.21g (yield 72%) of the objective compound (1-01) as a yellowish oil.

Synthesis example 2 Synthesis of Compound (2-01)

The synthesis of the compound (2-01) was carried out according to the above-mentioned production process. Specifically, the following operations are performed.

5.00g (33 mmol) of triethylene glycol, 14.39mL (112 mmol) of triethylamine and acetonitrile (50 mL) were introduced into a 200mL flask under argon atmosphere. After 7.97mL (103 mmol) of methanesulfonyl chloride was added dropwise at 0℃and stirred at room temperature for 1 hour. Next, 10mL of ethanol was added dropwise, and after the unreacted methanesulfonyl chloride was treated, it was filtered. The filtered reaction solution was washed 4 times with 50mL of methylene chloride and then with Na ₂ SO ₄ And (5) drying. The dried reaction solution was filtered and concentrated to obtain 8.21g (yield 81%) of intermediate (2-01-1) as an orange oil.

Next, 842mg (2.75 mmol) of intermediate (2-01-1) and K were introduced into a 100mL flask ₂ CO ₃ 950mg(6.87 mmol) and acetonitrile 15mL. After stirring at room temperature for 15 minutes, 735mg (8.258 mmol) of 3- (methylamino) -1-propanol was added dropwise. Stirring was carried out overnight while maintaining a temperature of 70 ℃. After the reaction solution was cooled, insoluble matter was removed by filtration. The filtrate was concentrated to give 720mg of crude product. The crude product was purified by column chromatography (NH silica gel 15g, developing solution 50% hexane/chloroform) to give 348mg (yield 43%) of intermediate (2-01-2) as a yellowish transparent oil.

To a 30mL eggplant-shaped flask were introduced 300mg (1.03 mmol) of the intermediate (2-01-2) and 10mL of methylene chloride, 770mg (2.56 mmol) of retinoic acid, 50mg (0.41 mmol) of 4-dimethylaminopyridine and 590mg (3.08 mmol) of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, and the mixture was stirred at room temperature overnight. Next, the reaction mixture was washed 2 times with 10mL of water and with Na ₂ SO ₄ And (5) drying. The dried reaction solution was filtered and concentrated to obtain 2.1g of a crude product. The crude product was purified by column chromatography (silica gel 40g, developing solution 50% hexane/chloroform and chloroform) to give 262mg (yield 29%) of the compound (2-01) as a dark orange oil.

(preparation of liposome containing DNA/peptide-core Complex entrapped with lipid Compound)

Using the vector DNA solution and the DNA condensation peptide, a core complex formed from the vector DNA-DNA condensation peptide is prepared. As vector DNA, a plasmid having integrated cytomegalovirus early promoter/enhancer, nluc gene, and transcription termination signal was used. As the DNA condensation peptide, mHP-1 (RQRRRR-YY-RQRRRR-GG-RRRRRRR: SEQ ID NO: 1) and mHP-2 (RRRRRRRRR-YY-RQRRRR-GG-RRRRRRRRRRR: SEQ ID NO: 2) were used as the DNA condensation peptides 1:3, and mixing the mixture in proportion.

100. Mu.l of a DNA condensation peptide solution (0.24 mg/ml, 10mM HEPES, pH 7.3) was dispensed into a microtube (Proteo Save SS (trade name) 1.5ml, manufactured by Sumitomo electric Co., ltd.). 200. Mu.l of a vector DNA solution (0.15 mg/ml, 10mM HEPES, pH 7.3) was added dropwise thereto and mixed while stirring the peptide solution thus dispensed with a vortex mixer (1,500 rpm) (MSV-3500 (trade name, manufactured by BIOSAN Co.).

The liposome of the inner core-spun complex is prepared by ethanol injection. 50. Mu.l of lipid solutions having the mixing ratios shown in Table 1 were dispensed into microtubes (Proteo Save SS (trade name) 1.5ml, manufactured by Sumitomo electric Co., ltd.). Here, as the compound for comparison, a compound represented by the formula (R-01) was used.

TABLE 1

TABLE 1

[ chemical 7]

While stirring the dispensed lipid solution with a vortex mixer, 50. Mu.l of the core complex was added dropwise and mixed. After the dropwise addition, 400. Mu.l of 10mM HEPES (pH 7.3) was gently added, thereby preparing a liposome containing the vector DNA. After 400. Mu.l of 10mM HEPES (pH 7.3) was added thereto, the mixture was gently mixed, and the mixture was subjected to centrifugation using an ultrafiltration spin column (PT-1014 (trade name), apro science, co., ltd.) to replace the buffer, and the concentrated solution was concentrated to prepare 100. Mu.l of liposomes (10 mM HEPES, pH 7.3) of the inner core complex.

[ evaluation of the amount of the inner DNA ]

For the obtained liposome, the amount of the entrapped DNA was measured. The measurement was performed by means of Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific). To 95. Mu.L of Tris-EDTA buffer containing 0.1% Triton-X100 (trade name), 5.0. Mu.L of liposome solution was added and gently suspended. After standing at room temperature for 30 minutes, 100. Mu.L of PicoGreen solution diluted 200-fold with Tris-EDTA buffer was added to the solution, followed by thorough mixing. After standing at room temperature for 5 minutes, using an enzyme-labeled instrument: mithras LB-940 (trade name, manufactured by Berthold Co.) measures the fluorescence intensity of the solution (excitation wavelength: 485nm, fluorescence wavelength: 530 nm).

DNA concentration was quantified using a standard curve made with known concentrations of lambda DNA. The amount of DNA in the liposome was calculated from the obtained values as the amount of DNA (μg DNA/mL) per 1mL of the solution. The results are shown in Table 2.

It was found that the amount of DNA entrapped in the liposome containing the compound of formula (1-01) was higher than that of the liposome containing the compound of formula (R-01). In addition, it was confirmed that when the compound of formula (2) was used as a lipid compound, the amount of the encapsulated DNA increased.

[ measurement of surface Charge of Liposome ]

The surface charge (zeta potential) of the liposome was measured by using a Zetasizer (Zetasizer Nano ZS (trade name, manufactured by Malvern Panalytical). 30. Mu.l of the liposome solution was dispensed into a zeta potential measuring cell (DTS-1070 (trade name), manufactured by Malvern Panalytical Co.) and 870. Mu.l of distilled water was added thereto and mixed, and the measuring cell was set in a Zetasizer to measure the zeta potential. The results obtained are shown in Table 2.

When the lipids forming the film using only DOPE as a neutral lipid (examples 1 to 4) and the lipids forming the film using DOPE as a cationic lipid (examples 1 to 6) were compared, it was found that zeta potential could be shifted further to the forward side in the latter case.

[ measurement of vector DNA introduction Using liposomes ]

The introduction of vector DNA into cells using liposomes can be quantified by expression of the Nluc gene on the vector DNA. Expression of the NLuc gene can be achieved by using a microplate reader: the amount of luminescence was measured and evaluated by infinite F200 (product name, manufactured by Tecan Co.). The cells used were Jurkat, MCF-7, and Huh-7 (from American Type Culture Collection). Seeding 1×10 into 96 well culture plates ⁶ After 100. Mu.L of the cell suspension in cell/mL, 1. Mu.L of the liposome solution shown in Table 1 was added. After addition, the cells were incubated at 37℃with 5% CO ₂ After 48 hours of incubation in an atmosphere incubator, the enzyme activity of NLuc was measured. The enzyme activity of NLuc was measured by a luminometer using NanoGlo Luciferase Assay System (product name, promega company) according to the manual attached to the kit. The results obtained are shown in FIG. 1 (Jurkat), FIG. 2 (MCF-7), and FIG. 3 (Huh-7).

It was confirmed that the lipid compound of formula (1) showed higher expression efficiency of the Nluc gene than (R-01) for comparison by selecting the lipid compound forming the membrane.

In addition, it was shown that, in terms of the expression efficiency of Nluc gene taken up into cells by liposomes, by combining the compound of formula (2) with the lipid compound of formula (1), it is easier to take up into adherent cells than into suspension cells. It is also known that cells having different proliferation patterns are directional by controlling surface charges with DOTAP or not.

TABLE 2

TABLE 2

Synthesis example 3 Synthesis of Compound (1-02)

The synthesis of the compound (1-02) was carried out according to the above-mentioned production process. Specifically, the following operations are performed.

Step 1

Magnesium (17.38 g,714.96mmol,4.40 eq.) diethyl ether (165 mL) and iodine (7 mg) were added to a 500mL flask under an argon atmosphere. After a few drops of 9-bromonon-1-ene (100.00 g,487.47mol,3.00 eq.) were added dropwise at room temperature, the mixture was refluxed for 2 hours. After aging overnight at room temperature, the Grignard reagent was transferred to a dropping funnel while washing with diethyl ether (40 mL). The grignard reagent was added dropwise to a 1000mL four-necked flask containing ethyl formate (12.04 g,162.49mmol,1.00 eq.) and diethyl ether (165 mL) at 0℃for 1.5 hours. After 1 hour of reaction at room temperature, acetone (100 mL) was added, followed by water (200 mL) and 10% sulfuric acid (267 mL) and separated. The aqueous layer was extracted with diethyl ether (300 mL) and dried over Na ₂ SO ₄ The organic layer was dried. The crude product (72.1 g) obtained by filtration and concentration was purified by column chromatography (silica gel 721g, development: hexane→3% ethyl acetate/97% hexane) to obtain 41.1g (yield 98%) of intermediate (1-02-1) as a white solid.

Step 2

Intermediate (1-02-1) (41.1 g,146.53mmol,1.00 eq.) was dissolved in methylene chloride (330 mL) under an argon atmosphere and then charged into a 1000mL flask, and triethylamine (59.31 g,586.12mmol, 4.00 eq.) and 4-dimethylaminopyridine (1.79 g,14.65mmol,0.10 eq.) were added . Methanesulfonyl chloride (33.57 g,293.06mmol,2.00 eq.) was added dropwise at-5 ℃. After stirring at room temperature for 1 hour, it was quenched with ice water (17.6 mL). Next, the mixture was washed with 1N hydrochloric acid (30 mL), water (300 mL), and saturated brine (300 mL), and then washed with Na ₂ SO ₄ And (5) drying. Filtration and concentration gave intermediate (1-02-2) 49.6g (yield 94%) as an orange oil.

Step 3

DMF (300 mL) and sodium cyanide (13.56 g,276.65mmol,2.00 eq.) were added to a 1000mL flask under argon atmosphere. 49.6g,138.32mmol,1.00 eq.) of intermediate (1-02-2) dissolved in DMF (200 mL) was added, warmed to 55℃and reacted overnight. The reaction mixture was allowed to return to room temperature, diluted with water (500 mL), and extracted with ethyl acetate (800 mL. Times.3). The extracted organic layer was washed with water (500 mL) and saturated brine (500 mL), and then dried over Na ₂ SO ₄ And (5) drying. The crude product (84.3 g) obtained by filtration and concentration was purified by column chromatography (silica gel 1012g, development: hexane → 5% ethyl acetate/95% hexane) to obtain 28.1g (yield 70%) of intermediate (1-02-3) as a pale yellow oil.

Step 4

To a 2000mL flask was added intermediate (1-02-3) (28.1 g,97.06mmol,1.00 eq.) and hexane (280 mL) under an argon atmosphere. 1M DIBAL-n-hexane (194.13 mL,194.13mmol,2.00 eq.) was added dropwise at-70℃and stirred at room temperature for 30 minutes. Ice-frozen to 0 ℃ and quenched with methanol (14 mL). Adding saturated NH into the reaction solution ₄ Aqueous Cl (1200 mL), stirred for 20 min, 10% H was added ₂ SO ₄ (450 mL) and separated. Then, the mixture was extracted with diethyl ether (500 mL. Times.2). The extracted organic layer was treated with saturated NaHCO ₃ Aqueous (500 mL) and saturated brine (500 mL) were washed with Na ₂ SO ₄ And (5) drying. Filtration and concentration gave intermediate (1-02-4) 25.3g (yield 89%) as a yellow oil.

Step 5

To a 1000mL flask was added intermediate (1-02-4) (25.3 g,86.5mmol,1.00 eq.) and methanol (253 mL). Sodium borohydride (1.16 g,30.27mmol,0.35 eq.) was added little by little at 0deg.C and stirred overnight at room temperature. Acetic acid (7 mL) was added to the reaction mixtureUntil a pH of 4 is reached. Water (160 mL) was added, extracted with dichloromethane (400 mL. Times.3) and the organic layer was extracted with Na ₂ SO ₄ And (5) drying. The crude product (30.3 g) obtained by filtration and concentration was purified by column chromatography (silica gel 304g, development: 5% ethyl acetate/95% hexane) to obtain 22.13g (yield 87%) of intermediate (1-02-5) as a pale yellow oil.

Step 6

Intermediate (1-02-5) (22.13 g,75.14mmol,1.00 eq.) was dissolved in methylene chloride (220 mL) in a 1000mL flask under argon atmosphere, and tetrabromomethane (29.90 g,90.17mmol,1.20 eq.) was added. Triphenylphosphine (29.56 g,112.71mmol,1.50 eq.) in dichloromethane (63 mL) was added dropwise at 0 ℃. After stirring at room temperature for 1 hour, the reaction solution was concentrated, and the obtained crude product (21.3 g) was purified by column chromatography (200 g of silica gel, development: hexane) to obtain 14.5g (yield 54%) of intermediate (1-02-6) as a colorless transparent oil.

Step 7

To a 200mL flask, ethanol (90 mL), 20% sodium ethoxide (solvent: ethanol) (50.55 g,148.57mmol,5.90 eq.) were added under an argon atmosphere and heated to 65 ℃. Subsequently, diethyl malonate (24.20 g,151.09mmol,6.00 eq.) and intermediate (1-02-6) (9 g,25.18mmol,1.00 eq.) were added and heated under reflux overnight. After the completion of the reaction, 1N hydrochloric acid (90 mL) was added at 10℃or lower, followed by quenching. The reaction was extracted with ethyl acetate (200 mL. Times.3), and extracted with saturated NaHCO ₃ The organic layer was washed with an aqueous solution (90 mL) and saturated brine (90 mL) successively, and then dried over Na ₂ SO ₄ And (5) drying. Filtration and concentration gave 7.31g (yield 66%) of intermediate (1-02-7) as an orange oil.

Step 8

To a 200mL flask was added intermediate (1-02-7) (7.31 g,16.74mmol,1.00 eq.), dimethyl sulfoxide (70 mL), sodium chloride (9.78 g,167.40 mmol), and heated under reflux overnight. After the completion of the reaction, the reaction mixture was concentrated, and the obtained crude product (21.3 g) was purified by column chromatography (200 g of silica gel: development: hexane → 2% ethyl acetate/98% hexane) to obtain 4.7g (yield 51%) of the intermediate (1-02-8) as a pale yellow oil.

Step 9

Lithium aluminum hydride (284 mg,19.34mmol,1.5 eq.) and THF (40 mL) were added to a 200mL four-necked flask under an argon atmosphere. Intermediate (1-02-8) (4.7 g,34.54mmol,1.00 eq.) dissolved in THF (40 mL) was added dropwise at 0 ℃ and reacted overnight at room temperature. Quench with ice cooled to 0 ℃, water (3.3 mL), 15% sodium hydroxide (0.8 mL). Ethyl acetate (50 mL) was added to the reaction mixture, and the mixture was filtered through fluoromagnesia, followed by washing the fluoromagnesia with ethyl acetate (100 mL). The filtrate was concentrated, and the obtained crude product (4.9 g) was purified by column chromatography (silica gel 50g, development: 5% ethyl acetate 95% hexane) to obtain 3.86g (yield 93%) of intermediate (1-02-9) as a colorless oil.

Step 10

Intermediate (1-02-9) (3.86 g,11.97mmol,1.00 eq.) was dissolved in methylene chloride (30 mL) under an argon atmosphere, and then charged into a 100mL flask, and triethylamine (4.84 g,47.87mmol,4.00 eq.) and 4-dimethylaminopyridine (146 mg,1.20mmol,0.10 eq.) were added. Methanesulfonyl chloride (2.74 g,23.93mmol,2.00 eq.) was added dropwise at-5 ℃. After stirring at room temperature for 1 hour, it was quenched with ice water (17.6 mL). Next, the mixture was washed with 1N hydrochloric acid (10 mL), water (30 mL), and saturated brine (30 mL), and then with Na ₂ SO ₄ And (5) drying. Filtration and concentration gave intermediate (1-02-10) 4.79g (yield 99%) as a brown oil.

11 th step

DMF (28 mL) and potassium cyanide (1.17 g,23.94mmol,2.00 eq.) were added to a 100mL flask under argon atmosphere. Intermediate (1-02-10) (4.79 g,11.97mmol,1.00 eq.) dissolved in DMF (20 mL) was added, warmed to 55℃and reacted overnight. The reaction mixture was allowed to return to room temperature, diluted with water (50 mL), and extracted with ethyl acetate (100 mL. Times.3). The extracted organic layer was washed with water (50 mL) and saturated brine (50 mL), and then dried over Na ₂ SO ₄ And (5) drying. The crude product (11.4 g) obtained by filtration and concentration was purified by column chromatography (silica gel 100g, development: hexane → 5% ethyl acetate/95% hexane) to obtain 3.6g (yield 90%) of intermediate (1-02-11) as a colorless oil.

Step 12

To a 100mL flask under argon atmosphere was added intermediate (1-02-11) (3.6 g,10.86mmol,1.00 eq.) and hexaneAlkane (36 mL). 1M DIBAL-n-hexane (21.71 mL,21.71mmol,2.00 eq.) was added dropwise at-70℃and stirred at room temperature for 30 minutes. Cooled to 0℃with ice and quenched with methanol (1.6 mL). Adding saturated NH into the reaction solution ₄ Aqueous Cl (150 mL) was stirred for 20 min, 10% sulfuric acid (50 mL) was added, and the mixture was separated. Then, the mixture was extracted with diethyl ether (50 mL. Times.2). The extracted organic layer was treated with saturated NaHCO ₃ Water (50 mL) and saturated brine (50 mL) were washed with Na ₂ SO ₄ And (5) drying. Filtration and concentration gave 3.2g (yield 88%) of intermediate (1-02-12) as a yellow oil.

Step 13

To a 100mL flask was added intermediate (1-02-12) (3.2 g,9.56mmol,1.00 eq.) and methanol (32 mL). Sodium borohydride (127 mg,3.35mmol,0.35 eq.) was added little by little at 0deg.C and stirred overnight at room temperature. Acetic acid (1 mL) was added to the reaction solution until pH4 was reached. Water (30 mL) was added, extracted with dichloromethane (30 mL. Times.3), and the organic layer was extracted with Na ₂ SO ₄ And (5) drying. The crude product (3.17 g) obtained by filtration and concentration was purified by column chromatography (silica gel 32g, development: 5% ethyl acetate/95% hexane) to obtain 1.12g (yield 35%) of intermediate (1-02-13) as a pale yellow oil.

Step 14

Intermediate (1-02-13) (1 g,2.97mmol,1.00 eq.) was dissolved in methylene chloride (10 mL) in a 30mL flask under argon atmosphere, and tetrabromomethane (1.18 g,3.57mmol,1.20 eq.) was added. Triphenylphosphine (1.17 g,4.46mmol,1.50 eq.) in dichloromethane (5 mL) was added dropwise at 0 ℃. After stirring at room temperature for 1 hour, the reaction solution was filtered and concentrated, and the obtained crude product (7 g) was purified by column chromatography (silica gel 70g, development: hexane) to obtain 1.12g (yield 94%) of intermediate (1-02-14) as a colorless transparent oil.

15 th step

To a 200mL flask was added intermediate (1-02-14) (1.12 g,2.80mmol,1.00 eq.) which was dissolved in dichloromethane (51 mL) and acetonitrile (51 mL), and ruthenium (III) chloride (29 mg,0.14mmol, ru=40%) was added. Sodium periodate (5.99 g,28.0mmol,10.00 eq.) dissolved in water (51 mL) was added dropwise below 10 ℃ and stirred overnight below 10 ℃. ReactionAfter completion, 51mL of water was added and the mixture was separated. To the organic layer was added saturated brine (50 mL), and 3% Na was added dropwise ₂ S aqueous solution until color change. 1N hydrochloric acid was added to the mixture until the mixture became acidic, and the mixture was separated. The organic layer was taken up with Na ₂ SO ₄ The crude product (5.24 g) obtained by filtration and concentration was purified by column chromatography (silica gel 50g, development: chloroform. Fwdarw.5% methanol/95% chloroform) to obtain 1.05g (yield 86%) of the intermediate (1-02-15) as a yellowish oil.

Step 16

In a 100mL flask, intermediate (1-02-15) (1.00 g,2.30mmol,1.00 eq.) was dissolved in dichloromethane (30 mL), and cis-2-nonen-1-ol (797 mg,5.60mmol,2.44 eq.) 4-dimethylaminopyridine (28 mg,0.23mmol,0.10 eq.) N, N-diisopropylethylamine (1.45 g,11.25mmol,4.90 eq.) was added. Then, 1- (3-dimethylaminopropyl) -3-ethyl-carbodiimide hydrochloride (1.07 g,5.603mmol,2.44 eq.) was added and stirred overnight at room temperature. After the reaction, it was diluted with dichloromethane (30 mL), diluted with water (30 mL), 1N hydrochloric acid (30 mL), and saturated NaHCO ₃ The organic layer was washed with water (30 mL) and saturated brine (30 mL) successively, and then dried over Na ₂ SO ₄ And (5) drying. The crude product (1.92 g) obtained by filtration and concentration was purified by column chromatography (silica gel 20g, development: hexane → 2% ethyl acetate/98% hexane) to obtain 77mg (yield 5%) of the intermediate (1-02-16) as a yellowish oil.

Step 17

In a 50mL autoclave, intermediate (1-02-16) (77 mg,0.11mmol,1.00 eq.) was dissolved with THF (3 mL), and 1-methylpiperazine (225 mg,2.25mmol,20.00 eq.) and potassium carbonate (56 mg,0.41mmol,3.6 eq.) were added. The temperature was raised to 55℃and the reaction was carried out for 6 days. After completion of the reaction, the reaction mixture was allowed to return to room temperature, diluted with methylene chloride (6 mL), and water (5 mL) was added thereto to separate the mixture. The aqueous layer was extracted with dichloromethane (6 mL. Times.3), and the combined organic layers were extracted with Na ₂ SO ₄ And (5) drying. The crude product (108 mg) obtained by filtration and concentration was purified by column chromatography (silica gel 1g, development: chloroform- > 10% methanol/90% chloroform) to obtain 54mg (yield 68%) of compound (1-02) as a dark yellow oil.

[ preparation of liposome containing lipid Compound-entrapped DNA/peptide core Complex ]

Using the vector DNA solution and the DNA condensation peptide, a core complex formed from the vector DNA-DNA condensation peptide is prepared. As vector DNA, a plasmid having integrated cytomegalovirus early promoter/enhancer, nluc gene, and transcription termination signal was used. As the DNA condensation peptide, mHP-1 (RQRRRR-YY-RQRRRR-GG-RRRRRRR: SEQ ID NO: 1) and mHP-2 (RRRRRRRRR-YY-RQRRRR-GG-RRRRRRRRRRR: SEQ ID NO: 2) were used as the DNA condensation peptides at a ratio of 4:1, and mixing the above materials in proportion.

100. Mu.l of a DNA condensation peptide solution (0.255 mg/ml, 10mM HEPES, pH 7.3) was dispensed into a microtube (Proteo Save SS (trade name) 1.5ml, manufactured by Sumitomo electric Co., ltd.). 200. Mu.l of a vector DNA solution (0.15 mg/ml, 10mM HEPES, pH 7.3) was added dropwise thereto and mixed while stirring the peptide solution thus dispensed with a vortex mixer (1,500 rpm) (MSV-3500 (trade name, manufactured by BIOSAN Co.).

The liposome of the inner core-spun complex is prepared by ethanol injection. 50. Mu.l of lipid solutions having the mixing ratios shown in Table 3 were dispensed into microtubes (Proteo Save SS (trade name) 1.5ml, manufactured by Sumitomo electric Co., ltd.). Reference examples 2-1 and 2-2 in table 3 were separate preparations using the same formulations as those of examples 1-2 and 1-4.

TABLE 3

TABLE 3 Table 3

While stirring the dispensed lipid solution with a vortex mixer, 50. Mu.l of the core complex was added dropwise and mixed. After the dropping, 900. Mu.l of 10mM HEPES (pH 7.3) was gently added to prepare liposomes of the inner core complex. Buffer exchange and concentration were performed by centrifugation using an ultrafiltration filter (CentriprepYM-50 (trade name, manufactured by Merck Co.) to prepare 600. Mu.l of liposome of the inner core-spun complex in 32 ml.

[ evaluation of the amount of the inner DNA ]

For the obtained liposome, the amount of the entrapped DNA was measured. The measurement was performed by means of Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific). To 99.5. Mu.L of Tris-EDTA buffer containing 0.1% Triton-X100 (trade name) and heparin sodium salt (Sigma-Aldrich Japan GK) was added 0.5. Mu.L of liposome solution, and gently suspended. After standing at room temperature for 30 minutes, 100. Mu.L of PicoGreen solution diluted 200-fold with Tris-EDTA buffer was added to the solution, followed by thorough mixing. After standing at room temperature for 5 minutes, using an enzyme-labeled instrument: mithras LB-940 (trade name, manufactured by Berthold Co.) measures the fluorescence intensity of the solution (excitation wavelength: 485nm, fluorescence wavelength: 530 nm).

DNA concentration was quantified using a standard curve made with known concentrations of lambda DNA. The amount of DNA in the liposome was calculated from the obtained values as the amount of DNA (μg DNA/mL) per 1mL of the solution. The results are shown in Table 4.

It was found that the liposome containing the compound of formula (1-02) showed the same amount of the entrapped DNA as the liposome containing the compound of formula (1-01). It was also confirmed that the amount of the DNA contained in the compound of formula (2-01) increases when the compound is combined.

[ measurement of surface Charge of Liposome ]

The surface charge (zeta potential) of the liposome was measured by using a Zetasizer (Zetasizer Nano ZS (trade name, manufactured by Malvern Panalytical). 10. Mu.l of the liposome solution was dispensed into a zeta potential measuring cell (DTS-1070 (trade name), manufactured by Malvern Panalytical Co.) and 890. Mu.l of distilled water was added thereto and mixed, and the measuring cell was set in a Zetasizer to measure the zeta potential. The results obtained are shown in Table 4. When comparing the case of using DOPE as a film-forming lipid (see example 2-2 and example 2-2) with the case of using DOPE as a film-forming lipid as a cationic lipid (see example 2-1 and example 2-1), it is understood that zeta potential can be shifted further to the forward side in the latter case.

[ measurement of vector DNA introduction Using liposomes ]

The introduction of vector DNA into cells using liposomes can be quantified by expression of the Nluc gene on the vector DNA. Expression of the NLuc gene can be achieved by using a microplate reader: the amount of luminescence was measured and evaluated by infinite F200 (product name, manufactured by Tecan Co.). The cells used were the human T cell leukemia cell line Jurkat (purchased from American Type Culture Collection) and human peripheral blood mononuclear cell PBMC (available from LONZA JAPAN Co.). Sowing 1×10 into 96-well culture plate ⁶ Cell suspension of Jurkat 100. Mu.L, 5X 10 cells/mL ⁶ After 100. Mu.L of the cell suspension of PBMC of cell/mL, the liposome solution shown in Table 1 was added so that the amount of DNA reached 0.8. Mu.g/well. After addition, the cells were incubated at 37℃with 5% CO ₂ After 48 hours of incubation in an atmosphere incubator, the enzyme activity of NLuc was measured. The enzyme activity of NLuc was measured by a luminometer using NanoGlo Luciferase Assay System (product name, promega) according to the manual attached to the kit. The results obtained are shown in FIG. 4 (Jurkat) and FIG. 5 (PBMC).

It was confirmed that DOTAP was shown to have higher expression efficiency in Jurkat cells than in Nluc gene of formula (1-01) when DOTAP was selected as the membrane-forming lipid compound in the lipid compound of formula (1-02). It was also confirmed that the lipid compounds of the formula (1-01) showed higher expression efficiency of Nluc gene than the lipid compounds of the formula (1-02) in PBMC cells when the lipid compounds of the formula (2-01) and DOPE were selected as the membrane-forming lipid compounds.

TABLE 4

TABLE 4 Table 4

[ lipid Compound-containing RNA-encapsulated liposomes ]

[ preparation of inner-coated RNA Liposome ]

Messenger RNA (mRNA) mRNA (OZ Biosciences) of green fluorescent protein GFP was used as a reporter gene. The RNA-encapsulated liposomes were prepared as follows: the GFP mRNA solution was added to a lipid solution having a mixing ratio shown in Table 5, and the mixture was suspended by blowing, and then 10mM HEPES (pH 7.3) was gently added thereto, and the solution was washed and concentrated by centrifugal ultrafiltration. Here, as the compound for comparison, a compound represented by the formula (R-01) was used. The comparative example R-3 was a separate preparation having the same formulation as that of the comparative example R-1.

TABLE 5

TABLE 5

[ measurement of the amount of the RNA contained in the inner bag ]

The amount of RNA entrapped in the liposomes was determined using the QuantiFluor RNASystem kit (Promega). The measurement was performed according to the manual attached to the kit. The measurement results of the amount of RNA inclusion are shown in Table 6. With respect to the liposome-encapsulated RNA prepared with the lipid solution of the compounding ratio of Table 5, no significant difference was seen in the amount of the encapsulated RNA of the liposome.

TABLE 6

TABLE 6

[ measurement of surface Charge of Liposome ]

The surface charge (zeta potential) of the RNA-encapsulated liposome was measured by Zetasizer (Zetasizer Nano ZS, malvern Panalytical). The liposome solution was placed in a zeta potential measuring cell (DTS-1070, manufactured by Malvern Panalytical Co.) and diluted and mixed with distilled water, and the cell was set at a predetermined position of a Zetasizer to measure the zeta potential. The amount of the RNA entrapped in the RNA-entrapped liposomes prepared in Table 5 and the average zeta potential are shown in Table 6.

[ measurement of RNA introduction Using liposomes ]

Introduction of RNA into cells using liposomes can be quantified by expression of GFP gene encoded by the RNA. The expression of GFP gene was evaluated by measuring the fluorescence by a flow cytometer (FACSVerse, BD Biosciences). The cells used were human T cell leukemia cell lines: jurkat (available from American Type Culture Collection). Jurkat cultured with TexMACS Medium (manufactured by Gibco Co.) was recovered by centrifugation to become 0.65X10 ⁷ The cells were suspended in fresh TexMACS. To the 48-well plate, 150. Mu.L each of the cell suspension and TexMACS was added to 1.0X10 ⁶ Cells/wells. Then, using the table 5 recordThe loaded RNA-encapsulated liposomes were added to the wells in such a manner that the amount of GFP RNA was 0.5. Mu.g/well, and the mixture was subjected to 5% CO at 37 ℃ ₂ Culturing under atmosphere. Here, as an RNA introduction method for comparison, a lipofection method using a Lipofectamine reagent (Lipofectamine 3000, manufactured by Invitrogen corporation) and an RNA introduction method using electroporation were performed. The introduction by the lipofection method was performed according to the manual attached to the reagent. For the introduction by electroporation, jurkat was recovered by centrifugation, washed by adding OptiMEM (Gibco), and then cells were recovered again by centrifugation. The recovered cells were then used as 1.0X10 ⁷ After suspending the cells/mL in OptiMEM, 0.5. Mu.g of GFP RNA was added to 100. Mu.L of the cell suspension, and transferred to a cell (Cuvette) electrode for electroporation. Electroporation was performed using CUY21 EDIT II (BEX) under the following conditions.

< conditions of perforation pulse (Pp) >)

Pp，225V；Pp on，2.5ms；Pp off，50.0ms

< drive pulse (Pd) Condition >)

Pd,20V; pd on,50.0ms; pd off,50.0ms;5, circulating; capacitor 1416.3. Mu.F

After culturing cells into which GFP RNA had been introduced for 48 hours, the cells were recovered, suspended in phosphate buffer PBS containing 1% BSA (Gibco), and the GFP fluorescence intensity (green fluorescence intensity) of the cells was measured by a flow cytometer. Fig. 6 shows the measurement results. The vertical axis of the graph is relative fluorescence intensity. Based on the results, the RNA-encapsulated liposomes prepared using the lipid solutions of examples 3-1 and 3-2 showed higher amounts than other RNA introduction methods, i.e., introduction by lipid transfection and introduction by electroporation. It was also found that the liposome containing the lipid solution of example 3-1 showed the highest amount of the liposome introduced.

[ measurement of vector DNA introduction Using liposomes ]

Liposomes containing inner core complexes of NLuc expressing plasmids were prepared as described in examples 1-3, 1-4 and 1-6 (examples 4-1, 4-2 and 4-3). The composition of the lipid solution constituting the liposome and the measurement result of the surface charge are shown in table 7 below.

TABLE 7

TABLE 7

The introduction of vector DNA into cells using liposomes can be quantified by expression of the Nluc gene on the vector DNA. Expression of the NLuc gene can be achieved by using a microplate reader: the amount of luminescence was measured and evaluated by infinite F200 (product name, manufactured by Tecan Co.). The cells used were Jurkat, MCF-7, and Huh-7 (from American Type Culture Collection). Sowing 1×10 in 96-well culture plate ⁶ After 100. Mu.L of the cell suspension per mL, 1. Mu.L of the liposome solution shown in Table 7 was added. After addition, the cells were incubated at 37℃with 5% CO ₂ After 48 hours of incubation in an atmosphere incubator, the enzyme activity of NLuc was measured. The enzyme activity of NLuc was measured using a luminometer according to the manual attached to the kit using NanoGlo Luciferase Assay System (product name, promega). The results obtained are shown in FIGS. 7 to 11.

FIGS. 7 to 9 are graphs of comparative examples 4-1 and 4-2, wherein FIG. 7 shows the expression efficiency of the Nluc gene in breast cancer cells (cell line MCF-7), FIG. 8 shows the expression efficiency of the Nluc gene in liver cancer cells (cell line Huh-7), and FIG. 9 shows the expression efficiency of the Nluc gene in leukemia cells (T-lymphocytes, cell line Jurkat). As shown in the results of FIGS. 7 to 9, by compounding the compound (2-01), the vector DNA contained in the liposome is more easily taken into adherent cells than into suspension cells.

FIGS. 10 and 11 are graphs of comparative examples 4-2 and 4-3, in which FIG. 10 shows the expression efficiency of the Nluc gene in breast cancer cells (cell line MCF-7) and FIG. 11 shows the expression efficiency of the Nluc gene in liver cancer cells (cell line Huh-7). From the results shown in FIGS. 10 and 11, it was found that, in a liposome containing a combination of the compound (1-01) and the compound (2-01), directivity to cells having different proliferation patterns can be imparted to the liposome by controlling the surface charge by using DOTAP or not.

As described above, although several embodiments of the present invention are illustrated, these embodiments are shown by way of example only and are not intended to limit the scope of the present invention. These novel embodiments may be implemented in various other modes, and various combinations, omissions, substitutions, modifications, and the like may be made without departing from the scope of the present invention. Such embodiments and modifications are included in the scope and spirit of the present invention, and are also included in the invention described in the claims and their equivalents.

Sequence listing

<110> Toshiba of Kagaku

<120> biodegradable compound, lipid particle, composition containing lipid particle, and kit

<130> 225966

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> mHP-1

<400> 1

Arg Gln Arg Gln Arg Tyr Tyr Arg Gln Arg Gln Arg Gly Gly Arg Arg

1 5 10 15

Arg Arg Arg Arg

20

<210> 2

<211> 21

<212> PRT

<213> artificial sequence

<220>

<223> mHP-2

<400> 2

Arg Arg Arg Arg Arg Arg Tyr Tyr Arg Gln Arg Gln Arg Gly Gly Arg

1 5 10 15

Arg Arg Arg Arg Arg

20

Claims (37)

1. A compound represented by the following formula (1),

Q-CHR ₂ (1)

in the method, in the process of the invention,

q is represented by the following formula (1-Q):

R ^Q1 ₂ N-(CR ^Q2 ₂ ) _q1 -NR ^Q1 -(CR ^Q2 ₂ ) _q2 -*(1-Q)

in the method, in the process of the invention,

R ^Q1 each independently of the other is an alkyl group,

R ^Q2 each independently is hydrogen or an alkyl group,

R ^Q1 r is R ^Q2 Any two of them may be bonded to form a nitrogen-containing alicyclic ring,

q1 is a number from 1 to 4,

q2 is a number from 0 to 4,

* Representation and-CHR ₂ Is used for the bonding position of the (c) and (d),

r is each independently C ₁₂ ～C ₂₄ Is represented by the following formula (1-R):

-L ^R1 -C(＝O)-O-L ^R2 (1-R)

in the method, in the process of the invention,

L ^R1 is an alkylene group, and is preferably an alkylene group,

L ^R2 represented by the following formula (1-R2):

-CH ₂ -CH＝CH-(CH ₂ ) _r2 -H(1-R2)

wherein r2 is a number of 1 to 10.

2. The compound of claim 1, wherein Q is any one of the structures shown below:

wherein, represents AND-CHR ₂ Is used for the bonding position of the substrate.

3. The compound of claim 1, wherein L ^R1 Represented by the following formula (1-R1),

-(CH ₂ ) _r1 - (1-R1)

wherein r1 is a number of 1 to 10.

4. A compound according to claim 3, wherein r1 is a number from 4 to 8.

5. The compound according to claim 1, wherein the longest molecular chain contained in R is 8 atoms or more.

6. The compound according to claim 1, which is represented by the following formulas (1-01), (1-02), (1-04) to (1-14), (1-16) and (1-18) to (1-21):

7. A lipid particle comprising a compound according to any one of claims 1 to 6.

8. The lipid particle according to claim 7, further comprising a compound,

the compound is represented by the following formula (2) and contains at least one biodegradable group selected from the group consisting of a carboxylic ester bond, a thiocarboxylic ester bond, a dithiocarboxylic acid bond, an amide bond, a urethane bond, a carboxydioxy bond and a urea bond in the structure,

P-[X-W-Y-W'-Z] ₂ (2)

in the method, in the process of the invention,

p is an alkyleneoxy group having 1 or more ether linkages in the main chain,

x is each independently a 2-valent linking group comprising a tertiary amine structure,

w is each independently C ₁ ～C ₆ An alkylene group,

y is each independently a 2-valent linking group selected from the group consisting of single bonds, ether bonds, carboxylic ester bonds, thiocarboxylic ester bonds, amide bonds, urethane bonds, and urea bonds,

w' are each independently a single bond or C ₁ ～C ₆ An alkylene group,

z is independently a fat-soluble vitamin residue, a sterol residue or C ₁₂ ～C ₂₂ Aliphatic hydrocarbon groups.

9. The lipid particle of claim 8, wherein P contains 3 to 8 carbons and 1 to 2 oxygens.

10. The lipid particle of claim 7 or 8, wherein each X is independently selected from the group consisting of methylimino, 1, 2-pyrrolidinediyl, and 1, 3-pyrrolidinediyl.

11. The lipid particle of claim 7 or 8, wherein Z is a fat-soluble vitamin residue or a sterol residue.

12. The lipid particle of claim 7 or 8, further comprising a membrane-forming lipid and an aggregation-reducing lipid.

13. The lipid particle of claim 12, wherein,

the membrane forming lipids are selected from the group consisting of:

1, 2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (DOPE),

1, 2-stearoyl-sn-glycerol-3-phosphatidylethanolamine (DSPE),

1, 2-dipalmitoyl-sn-glycerol-3-phosphatidylcholine (DPPC),

1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (POPC),

1, 2-di-O-octadecyl-3-trimethylammoniopropane (DOTMA),

1, 2-dioleoyl-3-dimethylammonium propane (DODAP),

1, 2-dimyristoyl-3-dimethylammonium propane (14:0 DAP),

1, 2-dipalmitoyl-3-dimethylammonium propane (16:0 DAP),

1, 2-distearoyl-3-dimethylammonium propane (18:0 DAP),

N- (4-carboxybenzyl) -N, N-dimethyl-2, 3-bis (oleoyloxy) propane (DOBAQ),

1, 2-dioleoyl-3-trimethylammoniopropane (DOTAP),

1, 2-dioleoyl-sn-glycerol-3-phosphorylcholine (DOPC),

1, 2-Dioleoyl-sn-glycerol-3-phosphorylcholine (DLPC),

1, 2-dioleoyl-sn-glycerol-3-phosphatidyl-L-serine (DOPS), and

cholesterol in the blood of a person,

the aggregation-reducing lipid is a polyethylene glycol (PEG) modified lipid.

14. The lipid particle of claim 7 or 8, further comprising an active agent.

15. The lipid particle of claim 14, wherein the active agent is a nucleic acid selected from the group consisting of a plasmid, an oligonucleotide, a polynucleotide, an siRNA, a micro RNA, DNA, mRNA, an aptamer, and a ribozyme.

16. The lipid particle of claim 14, wherein the active agent comprises a combination of at least one DNA and at least one RNA.

17. The lipid particle of claim 15, further comprising a compound that binds to a nucleic acid.

18. The lipid particle of claim 17, wherein the compound that binds to nucleic acid is an alkaline protein or an alkaline peptide.

19. The lipid particle of claim 17, wherein the compound that binds to nucleic acid is protamine or histone.

20. The lipid particle of claim 17, further comprising a compound that modulates the expression of a nucleic acid within a cell.

21. A composition comprising the lipid particle of any one of claims 7 to 20 and a carrier.

22. A composition for delivering an active agent to a cell, comprising the lipid particle of any one of claims 7 to 20 and a carrier.

23. The composition of claim 22, wherein the lipid particles contain both a compound of formula (1) and a compound of formula (2).

24. The composition of claim 23, wherein the molar ratio of the content of the compound of formula (2) to the content of the compound of formula (1) is less than 1.

25. The composition of claim 22 or 23, wherein the cell is a tumor cell.

26. The lipid particle according to any one of claims 7 to 20 for delivering an active agent to a cell.

27. The lipid particle according to claim 26, comprising both a compound of formula (1) and a compound of formula (2).

28. The lipid particle of claim 27, wherein the molar ratio of the content of the compound of formula (2) to the content of the compound of formula (1) is less than 1.

29. The lipid particle of claim 26 or 27, wherein the cell is a tumor cell.

30. A non-diagnostic and non-therapeutic method for delivering an active agent to cells of a subject other than a human, comprising: contacting the lipid particle of any one of claims 7 to 20 containing the active agent with a cell.

31. The method of claim 30, wherein the lipid particle contains both a compound of formula (1) and a compound of formula (2).

32. The method of claim 31, wherein the molar ratio of the content of the compound of formula (2) to the content of the compound of formula (1) is less than 1.

33. The method of claim 30, wherein the cell is a tumor cell.

34. Use of a lipid particle according to any one of claims 7 to 20 in the manufacture of a composition for delivering an active agent to a cell.

35. The use according to claim 34, wherein the lipid particles contain both a compound of formula (1) and a compound of formula (2).

36. Use according to claim 35, wherein the molar ratio of the content of the compound of formula (2) to the content of the compound of formula (1) is less than 1.

37. The use of claim 34 or 35, wherein the cell is a tumor cell.